Since the discovery of the importance of the mdr-1 gene product in multidrug resistance, most of the studies of the multidrug resistance phenotype have been performed at the nucleic acid level. We have established assays to examine drug resistance at the protein level by using flow cytometry to look at intact single cells. There are significant advantages to this approach, especially in probing the role of P-glycoprotein expression in innate and acquired multidrug resistance in clinical specimens. The assays we have developed allow us to measure P-glycoprotein expression and adriamycin content in each of thousands of cells, within hours of receiving a specimen. We have developed a two-parameter technique that allows us to correlate expression and adriamycin content in individual cells in a tumor specimen or cell line. Using this technique, we have been able to identify a subpopulation of innately resistant cells (high P-glycoprotein expression, low Adr content) in a CML-blast crisis cell line that was not detectable by previously available single parameter techniques. Conversely, we were able to detect a drug-sensitive subpopulation in a CML-blast crisis cell lines selected for Adr resistance and in MCF-7 cells transfected with the mdr-1 gene. In addition, we are working on flow cytometric analysis of other measures of drug resistance, including single cell detection of dihydrofolate reductase by fluoresceinated methotrexate and on the use of flow cytometry for the rapid screening of resistance reversal agents. We will use the techniques outlined to study drug resistance and its reversal in a variety of tumors. A project utilizing the unique multiparameter capabilities of flow analysis has been initiated to study the relationship of drug resistance to N-myc expression.